Bacillus thuringiensis subsp. israelensis has been used in the field for over twenty years without resistance development in any target insect. In contrast, mosquito resistance to Bacillus sphaericus has been observed in the field in many countries. This remarkable difference in the propensity to develop resistance to two mosquitocidal Bacillus strains is likely due to the presence of multiple toxins in B. thuringiensis subsp. israelensis. However, mosquitoes are able to rapidly develop resistance to individual toxins from this strain. A major reason for the apparent inability of mosquitoes to develop resistance to B. thuringiensis subsp. israelensis is the presence of cytolytic (Cyt) toxins in this and other mosquitocidal strains. The precise mechanism by which Cyt toxins interact with the insecticidal crystalline (Cry) toxins is not known, and therefore forms the basis for this proposal. We hypothesize that Cyt and Cry toxins interact in the membrane facilitating the formation of pores by either protein. In this proposal, our emphasis is on Cry11Aa and Cyt1Aa toxins of B. thuringiensis subsp. israelensis, which interact synergistically to enhance mosquitocidal activity of each toxin. The lack of resistance development is also due to two factors: the different molecular targets involved in the action of Cry and Cyt toxins, and secondly, the synergism between the Cyt and Cry toxins. Hence the objectives of the proposal are to first characterize the mechanism by which the Cry11Aa exerts its toxic effects, and the second is to understand the molecular basis of synergism between the Cry and Cyt toxins. In the first objective we will identify the domains involved in the receptor and toxin, identify the receptor(s), and finally isolate the receptor involved. Once the receptor is isolated we will use dsRNA-mediated gene silencing to determine its functional role as a Cry11A receptor. In the second objective our focus is on membrane binding of the toxins and the subsequent pore forming processes of both toxins. These two objectives are critical to our understanding of why no mosquito species have developed resistance to Bacillus thuringiensis subsp. israelensis. This project is also a joint proposal between three different investigators to best use the expertise of each laboratory.